Preparation Of Photoresponsive Camelliagenin Derivatives Cationic Liposome And Its Antibacterial Activity

Antibiotics are commonly applied in the therapy of pathogenic microorganism infection.However,the antimicrobial resistance can be easily acquired due to long-term abuse of antibiotics,which lead to the deficiency of therapeutic effects.Therefore,it has become currently urgent task how to find out a safe,high-effect and green antibiotic substitute.Based on separation and purification of Camelliagenin and its chemical structure modification and nanoencapsulation technology,a new antibacterial agent with the advantages of stability and light controlled release is designed and its antimicrobial mechanism is also revealed.The main research methods and conclusions were drawn as follows:(1)Through ultrafiltration-reverse osmosis combination with macroporous adsorption resin,the Camellia saponin with purity of 91.62%was acquired and then were successively hydrolyzed by acid and alkaline.The crude hydrolysis product was extracted with chloroform and recrystallization of ethanol/water mixed solvent to obtain the light yellow powder of Camelliagenin.Its structure was characterized by infrared(IR)and nuclear magnetism(NMR).(2)A total of 19 new series of Camelliagenin derivatives were synthesized by acylation reaction and their chemical structures were characterized by IR and NMR and HRMS.The growth inhibition ratio and minimum bactericidal concentration of the Camelliagenin derivatives against S.aureus and E.coli in vitro were respectively examined using the microdilution method,and biofilm formation was assayed by crystal violet staining.Combined with 3D-QSAR,the bioassay results indicated that its antibacterial activities were significantly enhanced.Notably,some new derivatives modified with aromatic ring or heterocyclic ring exhibited better antibacterial effect,furthermore,electron-withdrawing substituents at the meta or para position were favorable to increase the bioactivity.The effects of Camelliagenin derivatives on the bacterial morphology were observed with scanning electron microscope and transmission electron microscope,which showed obviously bacteria lysis.Subsequently,the effect on bacterial biofilm integrity was observed,which further confirmed its destructive effect on the cell membrane.The remarkable interactions were existed between Camelliagenin derivatives and mannitol dehydrogenase by molecular docking in favour of the inhibition of bacterial resistance.(3)SPE-MHA was synthesized via homogeneous reaction with phosphatidyl ethanolamine and maleic anhydride.CMC-g-SPE as a new carrier material was prepared via heterogeneous reaction between the carboxyl of SPE-MHA and the amino group of carboxymethyl chitosan(CMC),and then was purified by dialysis.The structure of CMC-g-SPE was verified by IR and~1H NMR,and the graft rate was 87.89%by elemental analysis.(4)A photoresponsive cationic liposome was prepared by reverse evaporation with CMC-g-SPE,soybean lecithin,cholesterol,Camelliagenin derivative(S-16)and pheophorbide.Based on encapsulation efficiency,average nanoparticle size and zeta potential as evaluation indexes,the preparation conditions of liposomes were optimized.The encapsulation efficiency,drug loading capacity,average nanoparticle size and zeta potential were respectively 83.52±1.53%,2.83±0.05%,189.23±1.11 nm and 18.80±2.37 mV under optimum conditions.The cationic liposomes had photoresponsiveness under 650 nm irradiation from the cumulative release curves in vitro.The increase of liposome phase-transition temperature and hydrophilicity dramatically improved the stability of liposomes through CMC-modified liposomes.(5)The antibacterial activity of cationic liposomes in vitro was evaluated using the microdilution method.The bioassay results indicated the antibacterial effect of photoresponsive cationic liposome could be significantly enhanced with 650 nm illumination and had synergistic effect with Camelliagenin derivatives through different antibacterial mechanisms.The erythrocytes hemolysis and HeLa cytotoxicity were assayed to evaluate the biosecurity of cationic liposome.It showed no hemolysis at concentrations<32?g/mL and no cytotoxicity at concentrations<4?g/m L,which suggested that the antibacterial activity of cationic liposomes was in the safe range.Through observing the interaction between cationic liposomes and bacteria and their effect on the bacterial morphology,photoresponsive cationic liposomes could destroy the structure of bacterial cell membrane and cell wall.The increased membrane permeability in the presence of photoresponsive cationic liposomes was further demonstrated its action on the cell membrane with the nucleic acid probe in the growth phases and stationary phase of bacteria.